1. Field of the Invention
The present invention relates to an optical waveguide type antibody chip and a method of measuring an antigen concentration using the chip.
2. Description of the Related Art
Conventionally, an enzyme-linked immunosorbent assay method (hereinafter, referred to as the ELISA method) has been put to practical use in the field of clinical laboratory testing, etc. as a method of measuring trace components by making use of a specific antigen-antibody reaction.
The ELISA method uses a resin plate having arranged therein 96 dents (wells), which is usually called a microplate. For example, in the sandwich ELISA method, a primary antibody is immobilized in each well according to its purpose. First, a specimen solution is poured into wells and an antibody immobilized on the plate is allowed to react with a measuring target substance in the specimen solution (hereinafter, referred to as primary reaction). Subsequently, the unreacted substances in the specimen solution are removed by cleaning. Thereafter, a solution of a secondary antibody marked with an enzyme is poured into wells and the secondary antibody is allowed to specifically react with the measuring target substance having reacted with the primary antibody (hereinafter, referred to as secondary reaction). The unreacted substances in the secondary specimen solution are removed by cleaning. A color reagent solution is poured into a well and subjected to an enzyme reaction to make the enzyme reaction product exhibit color. The absorbance is measured from the transmission light quantity of the well using a microplate reader. This absorbance is collated with a calibration curve prepared in advance to obtain the concentration of the measuring target substance.
For example, in order to form a diagnosis or pathosis of diabetes mellitus, the insulin concentration in blood is measured. Insulin is known to be a hormone secreted from the β cells of the pancreas and have a blood glucose lowering action. When the level of insulin is determined by the ELISA method, a specimen solution is poured into wells in a microplate on which an anti-insulin antibody is immobilized, and the anti-insulin antibody is caused to react with the insulin to obtain the insulin concentration according to the procedure described above.
The ELISA method using a microplate requires several dozen μL to about 100 μL of a specimen as a measuring specimen. At the minimum, 5 μL or more of a specimen is needed. Below this amount, the measuring sensitivity is too low, at only several thousands pg/mL.
As a different method, a sensor plate making use of an antigen-antibody reaction is disclosed in Jpn. Pat. Appln. KOKAI Publication No. 8-285851. This optical waveguide type sensor plate includes a glass plate, an optical waveguide layer constituted by a silicon nitride film formed on the substrate, incident-side and emitting-side gratings (refraction gratings) formed on both the ends of the optical waveguide layer, and an antibody immobilization layer formed on the optical waveguide layer. A prism can be used instead of a grating.
In the sensor plate having such a construction, the contact of a specimen solution containing an antigen on the antibody immobilization layer leads to an antigen-antibody reaction. When an antibody solution marked with a fluorescent dye is added to the antibody immobilization layer, an immune complex constituted by an antibody/antigen/fluorescent dye marked antibody is formed on the substrate. In this state, a laser beam is directed into the optical waveguide through the incident-side grating to generate an evanescent wave in the course of allowing the beam to propagate through the optical waveguide. The evanescent wave excites the fluorescent dye in the antibody immobilization layer on the optical waveguide layer and radiates fluorescence. The amount of fluorescence discharged from the fluorescent dye is detected by a light receiving element facing the antibody immobilization layer, whereby the antigen concentration in the specimen solution is measured.
Such evanescent wave is an electromagnetic wave generated only in the vicinity of the interface between an optical waveguide and an external layer when light is totally reflected at the interface. The known measuring methods using an evanescent wave also include, in addition to a method of marking with a fluorescent dye, a method of detecting changes in physical quantities of reflection light arising from the absorption of an evanescent wave in a pigment marked substance in a specimen (e.g., a secondary antibody marked with a pigment) (see Jpn. Pat. Appln. KOKOKU Publication No. 3-7270).
However, when the above sensor plate is used to measure the amount of insulin in blood plasma of, for example, a model rat (e.g., Zucker), the detection value is considerably lower than the actual value. In other words, it poses the problem of low detection reproducibility.